The possibility of preparing hybrid genes by gene technology has opened up new routes for the analysis of recombinant proteins. By linking the coding gene sequence of a desired protein to the coding gene sequence of a protein fragment having a high affinity for a ligand or antibody (affinity peptide), it is possible to purify desired recombinant proteins in the form of fusion proteins in one step using the affinity peptide.
Immobilized metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), is a specialized aspect of affinity chromatography. The principle behind IMAC lies in the fact that many transition metal ions, e.g., nickel, zinc and copper, can coordinate to the amino acids histidine, cysteine, and tryptophan via electron donor groups on the amino acid side chains. To utilize this interaction for chromatographic purposes, the metal ion is typically immobilized onto an insoluble support. This can be done by attaching a chelating group to the chromatographic matrix. Most importantly, to be useful, the metal of choice must have a higher affinity for the matrix than for the compounds to be purified.
In U.S. Pat. No. 4,569,794, Smith et al. disclose the preparation of a fusion protein containing a metal ion-affinity peptide linker and a biologically active polypeptide, expressing the fusion protein, and purifying it using immobilized metal ion chromatography. Because essentially any biologically active polypeptide could be used, this approach enabled the convenient expression and purification of essentially biologically active polypeptide by immobilized metal ion chromatography.
In U.S. Pat. Nos. 5,310,663 and 5,284,933, Dobeli et al. disclose a process for separating a biologically active polypeptide from impurities by producing the desired polypeptide as a fusion protein containing a metal ion-affinity peptide linker comprising 2 to 6 adjacent histidine residues. Although Dobeli et al.'s metal ion-affinity peptide provides greater metal affinity relative to certain of the sequences disclosed by Smith et al., there is some cautionary evidence that proteins containing His-tags may differ from their wild-type counterparts in dimerization/oligomerization properties. For example, Wu and Filutowicz present evidence that the biochemical properties of the pi(30.5) protein of plasmid R6K, a DNA binding protein, were fundamentally altered due to the presence of an N-terminal 6×His-tag. Wu, J. and Filutowicz, M., Acta Biochim. Pol., 46: 591-599 (1999). In addition, Rodriguez-Viciana et al. stated that V12 Ras proteins expressed as histidine-tagged fusion proteins exhibited poor biological activity. Rodriguez-Viciana, P., et al., Cell, 89: 457-67 (1997).
Antibodies specific for peptides may also be used to detect and purify proteins of interest. Typically, a protein of interest, or target protein, will be expressed in a host cell as a fusion protein comprising both the target protein and an antigenic domain, sometimes referred to as a tag, to which the specific antibodies will attach. The fusion protein can then be detected and purified according to well known methods in the art, such as, for example, Western blot and immunoprecipitation.
In U.S. Pat. No. 4,703,004, Hopp et al. disclose a hybrid polypeptide comprising an antigenic identification peptide and a peptide of interest, as well as nucleic acid sequences encoding the same. This hybrid polypeptide, once expressed, may be detected and purified by chromatographic techniques utilizing an immobilized antibody raised against the antigenic region of the identification polypeptide. This approach enabled the convenient expression and purification of any number of proteins by affinity techniques.
In U.S. Patent Publication No. US 2002-0045193 A1, Brizzard et. al. disclose an identification polypeptide comprising a tandem of antigenic domains and a linking sequence comprising a single cleavage sequence that is not present in the antigenic tandem. This approach provided a peptide tag having increased avidity as a result of the tandem of antigenic domains that could be cleaved from a fusion protein to produce a single target peptide product free of all antigenic domains of the tandem.
While both the metal ion-affinity peptide tags and the antibody based peptide tags have proved to be useful individually, there has yet to have been a tag having the properties of both practical metal affinity and practical antigenicity. Specifically, the metal ion-affinity peptides are generally not antigenic, as demonstrated by the use of antibodies raised against the polyhistidine metal ion-affinity peptides of U.S. Pat. Nos. 5,310,663 and 5,284,933. Methods of detecting and purifying such peptides based upon the use of anti-polyhistidine antibodies typically has resulted in non-specific binding or weak binding to the polyhistidine epitope. Likewise, methods of detecting and purifying hybrid polypeptides such as those disclosed in U.S. Pat. No. 4,703,004 by IMAC or MCAC have also proved to be less than ideal.